Figure 6.

CCN2 binds fibronectin and the fibronectin receptors integrin α4, integrin α5, integrin β1, and syndecan-4. (A) ¹²⁵I-CCN2 binds fibronectin. Wells of a 96 well plate were coated overnight at 4°C with 50 μg/ml fibronectin or 1% BSA in PBS or PBS and blocked for 1 h at room temperature with 1% BSA in PBS. ¹²⁵I-CCN2 (500,000 cpm/well) was added for 1 h, at room temperature. Wells were washed three times with 1% BSA in PBS and bound ¹²⁵I-CCN2 was measured with a scintillation counter. Values represent average ± SEM (N = 6). (B) Protein extracts (20 μg) from Ccn2+/+ and Ccn2-/- mouse embryonic fibroblasts (MEF) were subjected to Western blot analysis with an anti-CCN2 antibody as described in Materials and Methods. (C) Membrane preparations from Ccn2+/+ and Ccn2-/- mouse embryonic fibroblasts were immunoprecipated using protein A-Sepharose with rabbit anti-CCN2 antibody or rabbit IgG. Immunoprecipitates were subjected to Western blot analysis with anti-CCN2, anti-syndecan 4, anti-fibronectin, anti-endoglin, and anti-integrin α4, α5, and β1 antibodies, as indicated.